Reaction of dihydrofolate reductase with dansyl chloride. Chemical modification of a sensitive lysine residue and fluorometric studies of the dansylated enzyme

Dihydrofolate reductase from amethopterin-resistant Lactobacillus casei is virtually completely and irreversibly inactivated by relatively low concentrations of dansyl chloride. The complete inactivation can be correlated with the dansylation of a single lysine residue and ca. 90% quenching of protein fluorescence. This quenching phenomenon appears to be due, at least in part, to energy transfer from one or more excited state tryptophan residues to the covalently attached dansyl moiety. Under identical conditions lysine is not modified when the ternary complex of enzyme-NADPH-amethopterin is dansylated. The unreactive dansyl hydroxide protects the enzyme against dansyl chloride dependent inactivation and fluorescence studies indicate a single ligand binding site (KD = 1 x 10(-4) M). It is suggested that the dimethylaminonaphthyl moiety of dansyl chloride is directed to a hydrophobic region at or near the active center of the enzyme where a particularly susceptible lysine residue reacts to form a covalent bond with the reagent.